JP7717686B2 - Combined expression of chimeric CD3 fusion proteins and anti-CD3-based bispecific T cell activation elements - Google Patents

Description

BACKGROUND TECHNICAL FIELD This disclosure relates generally to immunotherapy, and in particular, this disclosure relates to the combined expression of chimeric CD3 fusion proteins and anti-CD3-based bispecific T cell activation elements.

Related Art In recent years, immunotherapy has achieved unprecedented success in complete remission rates for hematological malignancies. In 2017, two CAR-T products targeting CD19 were successfully marketed and approved for the treatment of acute leukemia and non-Hodgkin's lymphoma in children and adolescents, respectively.

However, immunotherapy for treating solid tumors has two major problems. On the one hand, the severe and even fatal clinical side effects associated with CAR-T therapy, mainly including cytokine release syndrome (CRS), macrophage activation syndrome, and neurotoxicity, remain a major risk to the clinical application of CAR-T therapy. Furthermore, CAR-T therapy has yet to demonstrate significant clinical efficacy in the clinical treatment of solid tumors.

WO2016070061 by Zhao et al. describes modified T cells expressing a bispecific antibody and a chimeric ligand-modified activating receptor (CLEAR). However, Zhao's disclosure is limited to the receptor/ligand targets PD1/PD-L1 and CD27/CD70, and the expression of PD1 or CD27 CLEAR.

WO 2016/054520 by Kim et al. describes effector cells expressing modified cell surface proteins and the use of the effector cells for the treatment of disease. One of their embodiments describes a combination therapy of effector cells with modified CD3e expression and a bispecific T cell-engaging antibody (BiTE). However, in Kim's method, CD3e and BiTE are not co-expressed on a single effector cell, and low-dose continuous infusion is required due to the short PK half-life and toxicity of BiTE. Independent administration of these effector cells and BiTE does not result in synergistic effects in the solid tumor microenvironment.

Therefore, there remains a need to develop therapeutic regimens for inhibiting solid tumors that have good clinical efficacy and few clinical side effects.

SUMMARY An object of the present disclosure is to provide a therapeutic regimen for inhibiting solid tumors that has good clinical efficacy and few clinical side effects.

A first aspect of the present disclosure provides DNA constructs encoding chimeric CD3 fusion proteins (e.g., chimeric CD3e fusion proteins) and anti-CD3-based bispecific T cell activation elements.

In a preferred embodiment, the chimeric CD3 is a fusion protein that includes one or more polypeptides that can be recognized by an anti-CD3 antibody, and, optionally, one or more of a transmembrane domain (TM), a costimulatory domain, and a CD3 signal activation domain, such as a CD3ζ domain.

In a preferred embodiment, the anti-CD3-based bispecific T cell activating element is a fusion protein comprising one or more tumor antigen recognition domains and one or more anti-CD3 antibody fragments, e.g., single domain antibody sequences (VHH), single chain antibody variable region sequences (scFv) and/or antigen-binding fragments (Fab) that target CD3. In a preferred embodiment, the chimeric CD3 fusion protein has the structure shown in Formula I:
L-EC-H-TM-C-CD3ζ (I)
In this formula,
L is absent or is a signal peptide sequence;
EC is a polypeptide binding domain from or derived from a CD3e protein that can be recognized by and binds to an anti-CD3 antibody;
The polypeptide binding domain is also referred to as the recognition binding domain of the anti-CD3 antibody;
The polypeptide binding domain is also referred to as a subunit of the recognition binding domain of the anti-CD3 antibody;
H is absent or is a linker or hinge region;
TM is the transmembrane domain;
C is absent or a costimulatory signaling molecule;
CD3ζ is a cytoplasmic signaling sequence that is absent or derived from CD3ζ;
Each "-" is independently a linker peptide or a peptide bond.

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, GM-CSFR (DNA SEQ ID NO: SEQ 1, AA SEQ ID NO: SEQ 2), CD4, CD137, or a combination thereof.

In another preferred embodiment, the polypeptide binding domain is the CD3e extracellular region or a portion thereof that can be recognized by an anti-CD3 antibody.

In another preferred embodiment, the anti-CD3 antibody is selected from the following group: scFV (single chain antibody), single domain antibody sequence (also known as nanobody or VHH), diabody, or variants thereof, or a combination thereof.

In another preferred embodiment, the anti-CD3 antibody clone comprises L2K (DNA SEQ ID NO: SEQ 19, AA SEQ ID NO: SEQ 20), UCHT1, OKT3, F6A, I2C, or a combination thereof. In another preferred embodiment, the polypeptide binding domain specifically recognizes and binds to an anti-CD3 antibody, which may be present as a segment of a bispecific antibody.

In another preferred embodiment, the EC comprises or consists of positions 1 to 104 of a wild-type or mutant CD3e protein, the amino acid sequence of which is set forth in SEQ ID NO: SEQ 4.

In another preferred embodiment, H is a linker or hinge region of a protein selected from the following group: CD8 (DNA SEQ ID NO: SEQ 5, AA SEQ ID NO: SEQ 6), CD28 (DNA SEQ ID NO: SEQ 57, AA SEQ ID NO: SEQ 58), CD137, or a combination thereof.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: CD28 (DNA SEQ ID NO: SEQ 59, AA SEQ ID NO: SEQ 60), CD3 epsilon, CD45, CD4, CD5, CD8 (DNA SEQ ID NO: SEQ 7, AA SEQ ID NO: SEQ 8), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, C is a protein costimulatory signal molecule selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

In another preferred embodiment, C comprises a 4-1BB-derived costimulatory signal molecule (DNA SEQ ID NO: SEQ 9, AA SEQ ID NO: SEQ 10) and/or a CD28-derived costimulatory signal molecule (DNA SEQ ID NO: SEQ 61, AA SEQ ID NO: SEQ 62).

In another preferred embodiment, CD3ζ is a cytoplasmic signaling sequence represented by AA SEQ ID NO:12.

In another preferred embodiment, the DNA construct is expressed in cis or fusion with a safety switch protein, and the protein capable of functioning as a safety switch includes inducible caspase 9 (iCasp9), CD19, CD20, EGFR, HER2, CD30, CD19, c-Met, claudin 18.2, or a combination thereof.

In another preferred embodiment, the anti-CD3-based bispecific T cell activation element (BiTA) has the structure shown in Formula II:
L'-T1-B1-B2-T2 (II)
In this formula,
L' is absent or is a signal peptide sequence;
T1 is absent or is a tag element;
B1 is a tumor antigen recognition region or a CD3 antigen recognition region;
B2 is a CD3 antigen-binding antibody fragment or a tumor antigen recognition region;
T2 is absent or is a tag element;
Each "-" is independently a linker peptide or a peptide bond.

In another preferred embodiment, L' is a signal peptide of a protein selected from the group consisting of CD8, GM-CSFR, CD4, CD137, or a combination thereof.

In another preferred embodiment, the tag element comprises a tag protein, a fluorescently labeled protein, or an enzyme-labeled protein.

In another preferred embodiment, the tag protein comprises a FLAG protein (DNA SEQ ID NO: SEQ 13, AA SEQ ID NO: SEQ 14) and a His protein (DNA SEQ ID NO: SEQ 35, AA SEQ ID NO: SEQ 36).

In another preferred embodiment, B1 is a tumor antigen recognition region and B2 is a CD3 antigen recognition region.

In another preferred embodiment, the tumor antigen recognition region comprises one or more receptor or ligand binding domains; an antibody fragment, including a single-domain antibody sequence (VHH) and/or a single-chain antibody variable region sequence (scFv); and/or a TCR sequence.

In another preferred embodiment, the tumor antigen is selected from the following group: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, DLL3, or a combination thereof.

In another preferred embodiment, the tumor antigen recognition region targets CAIX and/or HER2.

In another preferred embodiment, the tumor antigen recognition region is a VHH antibody targeting CAIX (DNA sequence number SEQ 17, AA sequence number SEQ 18).

In another preferred embodiment, the tumor antigen recognition region is a single-chain antibody targeting HER2 (DNA sequence number SEQ 65, AA sequence number SEQ 66).

In another preferred embodiment, the CD3 antigen-binding antibody fragment is a single-domain antibody sequence (VHH), a single-chain antibody variable region sequence (scFv), or an antigen-binding fragment (Fab) that targets CD3.

In another preferred embodiment, the BiTA is a secreted BiTA.
In another preferred embodiment, the BiTA targets CAIX (DNA SEQ ID NO: SEQ 21, AA SEQ ID NO: SEQ 22) or HER2 (DNA SEQ ID NO: SEQ 49, AA SEQ ID NO: SEQ 50).

In another preferred embodiment, the secreted BiTA may be autocrine and/or paracrine.

In another preferred embodiment, the secretory cell type of the secreted BiTA may be a T cell, an NK cell, a macrophage, a B cell, an erythrocyte, or a combination thereof.

In another preferred embodiment, the secretory cell type of the secreted BiTA is a T cell.
In another preferred embodiment, the BiTA is capable of binding to chimeric CD3e.

In another preferred embodiment, the BiTA is capable of binding to the T cell receptor (TCR) complex.

In another preferred embodiment, the TCR is derived from a T cell according to the fifth aspect and/or an unmodified T cell.

In another preferred embodiment, the nucleic acid molecule encoding the chimeric CD3 fusion protein and the nucleic acid molecule encoding the bispecific T cell activation element are provided separately. Advantageously, the nucleic acid molecule encoding the chimeric CD3 fusion protein and the nucleic acid molecule encoding the bispecific T cell activation element are co-expressed in the same immune cell.

A second aspect of the present invention provides a vector comprising the nucleic acid molecule.
In another preferred embodiment, the vector is selected from the group consisting of lentiviral, adenoviral, and retroviral vectors.

A third aspect of the present invention provides a genetically modified immune cell (e.g., a T cell) characterized in that it expresses the nucleic acid molecule described above. In an optional embodiment, the immune cell is modified to express a chimeric CD3 fusion protein and a bispecific T cell activating element, and the nucleic acid molecule encoding the chimeric CD3 fusion protein and the nucleic acid molecule encoding the bispecific T cell activating element are not provided on the same DNA construct.

In another preferred embodiment, the T cells are derived from a human or non-human mammal.
In another preferred embodiment, the T cell further comprises another chimeric antigen.

A fourth aspect of the present disclosure provides a composition comprising a fusion protein and a BiTA.

In another preferred embodiment, the fusion protein in the composition is located in the extracellular region of the T cell membrane.

In another preferred embodiment, the BiTA in the composition is an autocrine, paracrine, or exogenous BiTA.

In another preferred embodiment, the composition is expressed in the form of a fusion protein of structural formula I or structural formula II with a 2A protein, the structural formula being I-2A-II or II-2A-I, and the sequence of 2A includes one or a combination of T2A (DNA sequence number SEQ 23, AA sequence number SEQ 24), P2A, F2A, or E2A.

In another preferred embodiment, the I-2A-II construct is a sequence targeting CAIX (DNA SEQ ID NO: SEQ 25, AA SEQ ID NO: SEQ 26) or a sequence targeting HER2 (DNA SEQ ID NO: SEQ 49, AA SEQ ID NO: SEQ 50).

In another preferred embodiment, the II-2A-I construct is a sequence that targets CAIX or HER2 (DNA sequence number SEQ 67, AA sequence number SEQ 68).

In another preferred embodiment, the I-2A-II or II-2A-I structure is expressed in cis or fusion with a safety switch protein, and the protein capable of functioning as a safety switch includes inducible caspase 9 (iCasp9), CD19, CD20, EGFR, HER2, CD30, CD19, c-Met, claudin 18.2, or a combination thereof.

In another preferred embodiment, the composition is expressed in the form of a combination of an IRES sequence and a fusion protein of structural formula I or II, where the structural formula is I-IRES-II or II-IRES-I, and the IRES is an internal ribosome entry site of the nucleotide sequence.

In another preferred embodiment, the IRES functions to initiate amino acid translation of a downstream gene.

In another preferred embodiment, the I-IRES-II or II-IRES-I structure is expressed in cis or fusion with a safety switch protein, and the protein capable of functioning as a safety switch includes inducible caspase 9 (iCasp9), CD19, CD20, EGFR, HER2, CD30, CD19, c-Met, claudin 18.2, or a combination thereof.

A fifth aspect of the present disclosure provides a non-naturally occurring T cell population, wherein the T cell is present in the T cell population at a ratio C1 of 10% or greater based on the total number of T cells in the T cell population.

In another preferred embodiment, C1 is 10% or higher, preferably C1≧20%, and more preferably C1≧30%.

In another preferred embodiment, BiTA and/or BiTA-secreting T cells C2 are also present within the T cell population.

A sixth aspect of the present disclosure provides a composition comprising: (a) the genetically modified T cells and/or the T cell population described above; and (b) a pharmaceutically acceptable carrier, diluent, and/or excipient.

A seventh aspect of the present disclosure relates to the use of the above-described genetically modified T cells, T cell populations, and/or compositions in the preparation of a medicament for the prevention and/or treatment of cancer or tumors or for use in the methods described below.

An eighth aspect of the present disclosure provides a method for preventing or treating a disease, the method comprising administering an appropriate amount of the above-described genetically modified T cells, T cell population, and/or composition to a subject in need of treatment.

In another preferred embodiment, the disease is cancer or a tumor.
In another preferred embodiment, the tumor is selected from the following group: hematological tumors, solid tumors, and combinations thereof.

In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and combinations thereof.

In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, urinary tract tumor, thyroid cancer, and combinations thereof.

In another preferred embodiment, the solid tumor is selected from the following group: ovarian cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, liver cancer, endometrial cancer, or a combination thereof. In another preferred embodiment, the method further comprises administering an appropriate amount of a cytokine secreted by the stimulator cells or a drug compound or composition thereof to enhance immune cell responsiveness.

In certain embodiments, the method further comprises administering dasatinib to the subject.
In another preferred embodiment, the immune cells include the T cells, T cell populations and/or compositions described above, as well as endogenous T cells, NK cells, macrophages and B cells.

A ninth aspect of the present disclosure provides a method for reducing the toxicity of immune cells engineered with a chimeric CD3e fusion protein and a bispecific T cell activation element, the method comprising administering dasatinib; and the use of dasatinib in the preparation of a medicament for reducing the toxicity of immune cells engineered with a chimeric CD3e fusion protein and a bispecific T cell activation element.

It will be understood that within the scope of the present disclosure, various technical features of the present disclosure described above and various technical features specifically described below (e.g., in the Examples) may be combined with each other to form new or preferred technical solutions.

FIG. 1 shows the structure of CAB-T from the first experimental group and its control group. FIG. 1 shows the structure of CAB-T from the second experimental group and its control group. FIG. 10 shows the structure of CAB-T from the third experimental group and its control group. FIG. 1 shows the results of transduction frequency of T cells modified with the first group of constructs. FIG. 1 shows the results of transduction frequency of T cells modified with the second group of constructs. FIG. 1 shows the results of a transduction frequency assay of T cells modified with the third group of constructs. Figure 1 shows CAIX-CAB-T cytokine release assay (of constructs from the first experimental group). Figure 10: CAIX-CAB-T cytokine release assay (of constructs from the second experimental group). Figure 1 shows CAIX-CAB-T cell activation level assay (structures from the first experimental group). Figure 10: CAIX-CAB-T cell activation level assay (structures from the second experimental group). FIG. 10 shows HER2-CAB-T cell activation level assay (structures from the third experimental group). Figure 10: CAIX-CAB-T paracrine activated T cell level assay (of constructs from the second group). FIG. 10 shows HER2-CAB-T paracrine activated T cell level assay (of constructs from the third group). FIG. 10 shows immune checkpoint expression levels and cell differentiation phenotype analysis (of constructs from the second group) of CAIX-CAB-T and its control cells. FIG. 10 shows immune checkpoint expression levels and cell differentiation phenotype analysis of HER2-CAB-T and its control cells (of constructs from the third experimental group). FIG. 1 shows CAIX-CAB-T and its control cell-mediated tumoricidal ability assays (of constructs from the first experimental group). FIG. 1 shows CAIX-CAB-T and its control cell-mediated tumoricidal ability assays (of constructs from the second experimental group). FIG. 1 shows HER2-CAB-T and its control cell-mediated tumoricidal ability assays (of constructs from the third experimental group). FIG. 10 shows the structure of CAB-T from the fourth experimental group and its control group. FIG. 1 shows the results of transduction frequency of T cells modified with the fourth group of constructs. Figure 10 shows HER2-CAB-T cell activation level assay of constructs from (fourth experimental group). FIG. 1 shows the cell-mediated tumoricidal ability assay of HER2-CAB-T and its control (structures from the fourth experimental group). FIG. 1 shows CAIX ⁺ MDA-MB231 tumor growth inhibition assay in NCG-humanized mice at different doses of CAIX-CAB-T and its control cells. FIG. 23B shows the weight change of mice in the assay shown in FIG. 23A. Figure 23B shows images of tumors from mice in the assay shown in Figure 23A. FIG. 23B shows tumor weights in mice in the assays shown in FIG. 23A. FIG. 1 shows NCI-N87 tumor growth inhibition in M-NSG humanized mice treated with HER2-CAB-T and its control T cells. Figure 24B shows images of tumors from mice in the assay shown in Figure 24A. FIG. 24B shows the statistical tumor weights of mice in the assays shown in FIG. 24A. FIG. 1 shows that dasatinib inhibits IL-2 secretion from activated CAIX CAB-T at low concentrations. FIG. 1 shows that dasatinib inhibits IFN-γ secretion from activated CAIX CAB-T at low concentrations. FIG. 1 shows that dasatinib inhibits the lethal activity of CAIX CAB-T at low concentrations. FIG. 1 shows that dasatinib inhibits IL-2 secretion from activated HER2 CAB-T at low concentrations. FIG. 1 shows that dasatinib inhibits IFN-γ secretion from activated HER2 CAB-T at low dose concentrations. FIG. 1 shows that dasatinib inhibits the lethal activity of HER2 CAB-T at low concentrations. FIG. 1 shows a schematic diagram of the mechanism of action of CAB-T.

DETAILED DESCRIPTION The present disclosure relates to immunotherapeutic regimens for inhibiting tumors, particularly solid tumors, that involve the use of T cells expressing a chimeric CD3 fusion protein, preferably a chimeric CD3e fusion protein, together with an anti-CD3 antibody-based bispecific T cell activator (BiTA), which is similar to a bispecific T cell-inducing antibody (BiTE). The chimeric CD3 fusion protein and the BiTA, which has a CD3 antigen recognition site, bind to each other and function to activate T cells and target tumor cells expressing a tumor-associated antigen (TAA). The present disclosure also provides CAB constructs and CAB-modified T cells (CAB-T) that express the chimeric CD3 and anti-CD3 antibody-based bispecific T cell activator. BiTAs secreted by CAB-T cells simultaneously activate the endogenous TCR complexes of CAB-T cells and unmodified and modified T cells in tumor tissues, exerting the antitumor effect of CAB-T and mobilizing the antitumor effect of unmodified T cells, thereby ensuring the effectiveness of this CAB-T technology for clinical application. The chimeric CD3 constructs expressed by CAB-T and BiTAs work synergistically to exert their antitumor effects: activation of the chimeric CD3 element depends on BiTAs secreted by CAB-T, and secretion of BiTAs further stimulates CAB-T to secrete BiTAs by activating the chimeric CD3 element. Therefore, immune cell activation and antitumor effects are localized in the tumor microenvironment, ensuring the safety advantages of clinical application of this CAB-T technology.

Explanation of Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the term "about," when used in reference to a specifically recited value, means that the value may vary by up to 1% from the recited value. For example, as used herein, the phrase "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "contain" or "include" can be open-ended, semi-open-ended, or open-ended. In other words, the terms also include "consisting essentially of" or "consisting of."

The term "administering" refers to the physical introduction of a product of the present disclosure into a subject, for example, by injection or infusion, using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration.

The term "antibody" (Ab) is intended to include, but is not limited to, an immunoglobulin or antigen-binding fragment thereof that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domains that interact with an antigen.

It should be understood that the amino acid names herein are given using international English single-letter symbols, and that the English three-letter abbreviations corresponding to these amino acid names are Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V), respectively.

Chimeric Antigen Receptor (CAR)
The chimeric antigen receptor (CAR) structure is a fusion protein based on the intracellular segment domain of the TCR complex CD3ζ and the intracellular activator domain from the costimulatory signal CD28 or 4-1BB. Advantageously, T cells modified to express CARs can bind target antigens in an MHC-independent manner; thus, T cell activation does not depend on antigen presentation by MHC. This type of CAR is known as a second-generation CAR structure, and two CAR-T drugs approved in 2017 belong to this structural type.

T cell receptor (TCR)
The T cell receptor (TCR) is the most complex receptor in the human body, and its extensive signal transduction within T cells is determined by the interaction of six distinct receptor subunits. The two chains, TCRα and TCRβ, together recognize a complex composed of polypeptide-histocompatibility complexes. The subunits that transmit the TCR signal are collectively referred to as CD3 and include one heterodimer formed by CD3ε and CD3γ, one heterodimer formed by CD3ε and CD3δ, and one homodimer of CD3ζ. All subunits of the TCR, except for CD3ζ, are type I transmembrane proteins and contain immunoglobulin domains. The four distinct CD3 subunits in the TCR receptor complex contain a total of 10 immunoreceptor tyrosine-based activation motifs (ITAMs), which can accept a total of 20 tyrosine phosphate groups upon activation of the TCR receptor complex. Transgenic mouse experiments have shown that alterations in the proline-rich region of the intracellular segment of CD3ε or alterations in the conformation of CD3ε play crucial regulatory roles in the delivery of intact TCRs. It has been demonstrated that TCR activity can be modulated by ligand binding to TCRαβ, stabilizing the arrangement of CD3 subunits, ligand-independent TCR oligomerization, and cholesterol binding.

TRuC™, a novel T cell therapy platform developed in-house by ^TCR2 , is a chimeric antigen receptor (TCR) consisting of an antibody-based target antigen recognition sequence and a TCR receptor subunit. The TRuC construct can reprogram the entire TCR complex to recognize tumor antigens. Unlike the CAR construct, the TRuC construct can be incorporated into the TCR complex to exert its function. TRuC-T possesses the same tumoricidal activity as second-generation CAR-T. In addition, TRuC-T cells release cytokines at significantly lower levels than CAR-T cells due to the absence of additional costimulatory signaling domains (CD28 or 4-1BB). TRuC-T demonstrates antitumor activity in both hematological and solid tumor xenograft models. Additionally, TRuC-T demonstrates stronger antitumor activity compared to CAR-T in multiple tumor models.

T cell antigen conjugate (TAC)
Triumvira's TAC (T-cell antigen conjugate) technology platform can induce stronger antitumor responses than CAR-T with lower toxicity by modulating the endogenous TCR of T cells. The TAC structure consists of three parts: 1. an extracellular antigen-binding domain, 2. a TCR-engaging domain of a CD3 single-chain antibody, and 3. a CD4/CD8 co-receptor binding domain. Preclinical experiments have demonstrated that TAC-T technology can specifically bind to tumor cells and induce cytotoxicity, and that activation of TAC-T is similar to that of normal T cells, thus preventing the production of multiple cytokines. In mouse tumor xenograft models, TAC-T exhibits better activity than CAR-T against both solid and hematological tumors. In addition, TAC-T has a high ability to infiltrate the tumor microenvironment of solid tumors.

Bispecific T cell inducing antibody (BiTE)
Blinatumomab, a bispecific T cell-engaging antibody (BiTE) drug targeting CD19 and developed by Amgen, USA, was approved by the FDA for the clinical treatment of acute leukemia in 2014. This antibody consists of two parts: an scFv that recognizes the CD19 antigen and an scFv that recognizes the TCR complex (CD3e). Upon recognizing the target antigen CD19 in tumor cells, the BiTE antibody utilizes the anti-CD3 scFv portion to induce oligomerization of the T cell's endogenous TCR, thereby activating the T cell and inducing tumor lethality. The method by which BiTE treats tumors is similar to that of TRuC and TAC technologies, all of which cause the activation of endogenous TCR in T cells. Theoretically, these three have equivalent abilities to activate endogenous TCR signals, and all may be highly useful for mobilizing and redirecting T cells to treat solid tumors. However, due to the poor safety profile resulting from systemic administration of BiTE drugs, their extremely short half-lives in vivo, and the like, the use of BiTEs has not yet been demonstrated to achieve the desired efficacy in the clinical treatment of solid tumors.

Bispecific T Cell Activator (BiTA) Structures The terms "bispecific T cell activator structure,""bispecific T cell activating element,""BiTA,""bispecific T cell activator," and "-BiTA" as referred to herein refer to anti-CD3-based bispecific T cell activator structures that comprise two portions: (i) one or more tumor antigen-recognizing regions, such as receptor or ligand-binding domains, that are antibody fragments comprising a single domain antibody sequence (VHH), a single chain antibody variable region sequence (scFv), an antigen-binding fragment (Fab), and/or a T cell receptor (TCR) sequence that recognize a tumor antigen, and (ii) one or more CD3 antigen-recognizing regions, such as antibody fragments comprising a single domain antibody sequence (VHH), a single chain antibody variable region sequence, or an antigen-binding fragment (Fab) that targets CD3.

In another preferred embodiment, the anti-CD3-based bispecific T cell activating element has the structure shown in Formula II:
L'-T1-B1-B2-T2 (II)
In this formula,
L' is absent or is a signal peptide sequence derived from a protein selected from the group consisting of CD8, GM-CSFR, CD4, CD137, or a combination thereof.

T1 is absent or is a tag element, which optionally comprises a tag protein, a fluorescently labeled protein, or an enzyme-labeled protein. Preferably, the tag protein comprises a FLAG protein (DNA SEQ ID NO: SEQ 13, AA SEQ ID NO: SEQ 14) and a His protein (DNA SEQ ID NO: SEQ 35, AA SEQ ID NO: SEQ 36).

B1 is a tumor antigen recognition region, which optionally includes a receptor or ligand binding domain, a single domain antibody sequence (VHH), and/or a single chain antibody variable region sequence (scFv), and/or an antibody Fab, and/or a T cell receptor (TCR) sequence, which/these can recognize a tumor antigen selected from the following group: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, DLL3, or a combination thereof. Preferably, the tumor antigen recognition region B1 targets the tumor antigen CAIX and/or HER2.

In another preferred embodiment, the tumor antigen recognition region B1 is a VHH antibody targeting CAIX (DNA sequence number SEQ 17, AA sequence number SEQ 18).

In another preferred embodiment, the tumor antigen recognition region B1 is a single-chain antibody derived from trastuzumab that targets HER2 (DNA sequence number SEQ 65, AA sequence number SEQ 66).

B2 is a CD3 antigen recognition region, optionally a single domain antibody sequence (VHH) and/or antibody Fab and/or single chain antibody variable region sequence (scFv) targeting CD3. In a preferred embodiment, the CD3 antigen-binding antibody fragment may be derived from a CD3 Ab clone such as L2K, UCHT, OKT3, F6A, or SP34.

If desired, the positions of B1 and B2 may be reversed.
T2 is absent or a tag element, which optionally comprises a tag protein, a fluorescently labeled protein, or an enzyme-labeled protein. Preferably, the tag protein comprises a FLAG protein (DNA SEQ ID NO: SEQ 13, AA SEQ ID NO: SEQ 14) and a His protein (DNA SEQ ID NO: SEQ 35, AA SEQ ID NO: SEQ 36).

Each "-" is independently a linker peptide or a peptide bond.
CD3e Protein As mentioned herein, "CD3e protein" and "CD3e" both refer to the human CD3e protein.

As used herein, the "extracellular region of the CD3e protein" refers to amino acids 1 to 104 of the CD3e protein sequence exemplified by SEQ ID NO:4.

Protein sequences include amino acid sequences that have 60% or more, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequence.

Chimeric CD3e Fusion Proteins "Chimeric CD3e fusion protein,""CD3e fusion protein," and "chimeric CD3e protein," as referred to herein, refer to a fusion protein expressed in a T cell having the structure shown in Formula I:
L-EC-H-TM-C-CD3ζ (I)
In this formula,
each "-" is independently a linker peptide or a peptide bond;
L is an optional signal peptide sequence;
EC is a polypeptide binding domain from or derived from a CD3e protein that can be recognized by and binds to an anti-CD3 antibody;
The polypeptide binding domain is also referred to as the recognition binding domain of the anti-CD3 antibody;
The polypeptide binding domain is also referred to as a subunit of the recognition binding domain of the anti-CD3 antibody;
H is an optional linker or hinge region;
TM is the transmembrane domain;
C is absent or a costimulatory signal molecule;
CD3ζ is a cytoplasmic signaling sequence that is absent or derived from CD3ζ.

In another preferred embodiment, the polypeptide binding domain is the CD3e extracellular region exemplified by SEQ ID NO:4, or a portion thereof that can be recognized by an anti-CD3 antibody.

In another preferred embodiment, the anti-CD3 antibody is selected from the following group: scFV (single-chain antibody), single-domain antibody sequence (also known as nanobody), diabody, antibody Fab or variant thereof, or a combination thereof.

In another preferred embodiment, the polypeptide binding domain specifically recognizes and binds to an anti-CD3 antibody, which may be present as a segment of a bispecific antibody.

In another preferred embodiment, the hinge region is a CD8 hinge, the amino acid sequence of which is SEQ ID NO: 3.

In another preferred embodiment, the transmembrane domain is CD8 TM, the amino acid sequence of which is SEQ ID NO: 8.

Chimeric CD3 and anti-CD3 based bispecific T cell activator modified T cells, CAB-T
As referred to herein, "chimeric CD3 and anti-CD3 based bispecific T cell activator engineered T cells,""CAB-Tcells,""CAB-Ttechnology,""CABstructure,""CAB-T,""-CAB-T," and "-CAB" refer to T cells engineered to co-express both a chimeric CD3 fusion protein and an anti-CD3 based bispecific T cell activator. Preferably, the modified T cells comprise a construct having the following structure: (i) a chimeric CD3e in a CAB structure comprising at least the following four components: a CD3e extracellular domain, a CD8 hinge and transmembrane domain, a 4-1BB intracellular domain, and a CD3 intracellular domain; (ii) an anti-CD3-based bispecific T cell activator (BiTA) comprising at least the following two moieties: a receptor or ligand binding domain that is an antibody fragment comprising a single domain antibody sequence (VHH), an antibody Fab fragment, a single-chain antibody variable region sequence (scFv), or a T cell receptor (TCR) sequence that recognizes and binds to a tumor antigen, and a variable region sequence that recognizes the CD3 antigen in the TCR complex.

As mentioned above, the amino acid sequence of the extracellular region of CD3e is shown in SEQ ID NO: 4, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 10, and the amino acid sequence of CD3ζ is shown in SEQ ID NO: 12.

Other preferred synthetic gene sequences are shown in Table 1 below:

CAIX
CAIX is a transmembrane protein expressed in various solid tumor cells. Its main function is to maintain intracellular pH homeostasis under hypoxic conditions commonly found in solid tumors. CAIX expression in tumor cells is considered a marker protein for hypoxic tumor environments and poor patient prognosis. Common types of tumors that express CAIX include cervical cancer, kidney cancer, brain cancer, head and neck cancer, esophageal cancer, intestinal cancer, breast cancer, ovarian cancer, endometrial cancer, bladder cancer, and the like. In normal tissues, CAIX is mainly expressed in bile duct and small intestinal epithelial cells and gastric epithelial cells, but unlike tumor cells, CAIX expressed in normal tissues is mainly localized in the cytoplasm. Therefore, CAIX is an ideal therapeutic target for targeted therapy, including cell therapy.

HER2
HER2 is one of the most studied targets for tumor immunotherapy and is commonly expressed in tissues such as breast cancer, gastric cancer, colon cancer, cervical cancer, endometrial cancer, urothelial cancer, ovarian cancer, and lung cancer. Trastuzumab, a monoclonal antibody drug targeting HER2, has greatly improved the quality of life and prolonged the lifespan of patients with HER2-positive breast cancer, but there are still many patients with HER2-positive tumors that are ineffective or develop resistance to trastuzumab. Therefore, there is still a great market demand for the development of new therapies targeting HER2. Currently, there are reports of CAR-T drugs targeting HER2. Among them, Steven A. Rosenberg reported severe toxicity and side effects in clinical trials of third-generation CAR-T drugs targeting HER2, which caused respiratory distress and severe immune cell infiltration in the lungs, leading to patient death. Therefore, during the development stage of drugs targeting HER2 cells, such drugs must be designed to emphasize the safety of the drug.

Compositions The present disclosure provides compositions or formulations comprising T cells engineered to co-express both a chimeric CD3 fusion protein and an anti-CD3-based bispecific T cell activator (i.e., CAB-T cells), together with a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the composition is a liquid formulation. Preferably, the composition is an injectable formulation. Preferably, the concentration of CAB-T cells in the composition is 1 x ¹⁰ to 1 x ¹⁰ cells/ml, more preferably 1 x ¹⁰ to 1 x ¹⁰ cells/ml.

In one embodiment, the composition may include a buffer, such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate, such as glucose, mannose, sucrose, or dextran, and mannitol; a protein; a polypeptide or an amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present disclosure are preferably formulated for intravenous administration.

Therapeutic Applications The present disclosure encompasses therapeutic applications of T cells engineered to co-express both a chimeric CD3 fusion protein and an anti-CD3-based bispecific T cell activator (i.e., CAB-T cells). Cells transduced with vectors containing the nucleic acid constructs of the present disclosure can target tumor cell markers, while autocrine or paracrine BiTAs (secreted by the engineered T cells) can synergistically activate T cells and trigger T cell immune responses, thereby significantly improving their lethal efficiency against tumor cells.

Accordingly, the present disclosure also provides a method for stimulating a T cell-mediated immune response in a target cell population or tissue in a mammal, the method comprising administering CAB-T cells.

Cancers that can be treated include vascularized tumors as well as non- or substantially non-vascularized tumors. Cancers can include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or solid tumors. Types of cancer that can be treated with the nucleic acid constructs and modified T cells of the present disclosure include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant diseases such as sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (blood-borne) cancers include leukemia, including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, granulocytic-monocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell type that forms them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

The CAB-modified T cells of the present disclosure can also be used as a vaccine for ex vivo immunization and/or in vivo therapy in a mammal. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following is performed in vitro prior to administering the cells to the mammal: i) expanding the cells, ii) introducing a nucleic acid encoding the CAB into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal, preferably a human, and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAB disclosed herein. The CAB-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be human, and the CAB-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

In addition to the use of cell-based vaccines for ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The present disclosure provides a method for treating a tumor, the method comprising administering to a subject a therapeutically effective amount of CAB-modified T cells of the present disclosure.

The CAB-modified T cells of the present disclosure can be administered in the form of a pharmaceutical composition, alone or in combination with a diluent and/or other components, such as IL-2, IL-17, or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present disclosure can include a population of target cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, and mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but appropriate dosages can be determined through clinical trials.

When referring to an "immunologically effective amount,""antitumor effective amount,""tumor suppression effective amount," or "therapeutic amount," the exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account the patient's (subject's) age, weight, tumor size, and extent of infection or metastasis, as well as individual differences in the condition. It is generally indicated that pharmaceutical compositions comprising the T cells described herein can be administered at a dose of ^{10 to 10} cells/kg body weight, preferably 10 to ¹⁰ cells/kg body weight (including all integer values within these ranges). Multiple doses of these T cell compositions can also be administered. Cells can be administered using injection techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for an individual patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

The compositions can be administered to a subject by any conventional method, including by inhalation, injection, oral administration, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intrathecally, intramuscularly, intravenously (i.v.), or intraperitoneally. In one embodiment, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present disclosure are preferably administered by i.v. injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

In certain embodiments of the present disclosure, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of related therapeutic approaches, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for patients with MS, or efalizumab treatment for patients with psoriasis, or other treatments for patients with PML. In further embodiments, the T cells of the present disclosure can be used in combination with chemotherapy, radiation, immunosuppressants, e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutics. In further embodiments, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) a bone marrow transplant, a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an injection of the expanded immune cells of the present disclosure after transplant. In a further embodiment, the expanded cells are administered before or after surgery.

The dosages of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dosage rates administered to humans can be carried out according to art-accepted practices. Typically, 1× ¹⁰ to 1× ¹⁰ modified T cells of the present disclosure can be administered to a patient per treatment or course of treatment, e.g., by intravenous reinfusion.

The technical solutions of the present disclosure have the following beneficial effects:
1. The present disclosure provides an immunotherapeutic regimen for inhibiting tumors, particularly solid tumors, that combines T cells expressing a BiTA and a chimeric CD3 fusion protein. The chimeric CD3 fusion protein and the BiTA bind to each other and function to activate T cells and target tumor cells.

2. The present disclosure also provides CAB technology, in which CAB-T cells target tumor tissues through chimeric expression of CD3 and BiTA in T cells. The BiTA secreted by the CAB-T cells simultaneously activates the CAB-T cells and the endogenous TCR complexes of unmodified T cells in tumor tissues, thereby exerting the anti-tumor effect of the CAB-T itself and mobilizing the anti-tumor effect of unmodified T cells, thereby ensuring the efficacy of clinical application of CAB-T.

3. Because the activation of chimeric CD3 depends on BiTA secreted by CAB-T, a small amount of BiTA released by CAB-T in tumor tissue can stimulate CAB-T to release more BiTA in the local tumor microenvironment, which advantageously ensures the safety of clinical application of CAB-T.

4. CAB-T can achieve better activation in solid tumor tissue, maximize antitumor efficacy at the tumor site, and achieve safety and efficacy similar to that of local administration within the tumor, giving it significant advantages and potential compared to second-generation CAR-T in the clinical treatment of solid tumors.

The present disclosure is further illustrated below with specific examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions as described, for example, by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Example 1 Design of CAB and Its Control Structures 1.1 Structural Design of CD3e-BBζ, the First CAIX-BiTA, the First CAIX-CAB, and CAIX-TRuC in the Control Group To verify the antitumor activity of CAB-T, we first designed four structures in the first group: CD3e-BBζ (DNA SEQ ID NO: SEQ 15, AA SEQ ID NO: SEQ 16), CAIX-BiTA (DNA SEQ ID NO: SEQ 21, AA SEQ ID NO: SEQ 22), CAIX-CAB (DNA SEQ ID NO: SEQ 25, AA SEQ ID NO: SEQ 26), and CAIX-TRuC (DNA SEQ ID NO: SEQ 31, AA SEQ ID NO: SEQ 32) using nanobodies (VHHs) targeting CAIX in the experimental group. To distinguish them from the second group of CAIX-CAB constructs described later, we named this group of CAIX-BiTAs and CAIX-CABs the first CAIX-BiTAs and first CAIX-CABs, respectively. Among these, the first CAIX-BiTA is a tagless BiTA, the first CAIX-CAB contains CD3e-BBζ and CAB constructs in which the BiTA is not labeled, and the CAIX-TRuC is a control construct that uses TCR ² Therapeutics' platform technology. The specific structures of the above constructs are shown in Figure 1.

1.2 Structural Design of tERBB2, CD3e-BBζ, CAIX-BiTA, CAIX-CAB, CAIX-ζ, CAIX-BBζ, and CAIX-28ζ in Experimental Groups of Nanobodies (VHHs) Targeting CAIX In another experimental group, the inventors used VHHs targeting CAIX to engineer truncated ERBB2 (tERBB2, which was used as a negative control and contains the four extracellular domains, transmembrane region, and FLAG tag of ERBB2) (DNA SEQ ID NO: SEQ 33, AA SEQ ID NO: SEQ 34), CD3e-BBζ (DNA SEQ ID NO: SEQ 15, AA SEQ ID NO: SEQ 16), CAIX-BiTA (DNA SEQ ID NO: SEQ 37, AA SEQ ID NO: SEQ 38), CAIX-CAB (DNA SEQ ID NO: SEQ 19), and CAIX-28ζ. A second group of structures was designed, including seven structures in total: CAIX-ζ (a first-generation CAR structure targeting CAIX) (DNA SEQ ID NO: SEQ 41, AA SEQ ID NO: SEQ 42), CAIX-BBζ (a second-generation CAR structure containing a 4-1BB costimulatory domain) (DNA SEQ ID NO: SEQ 45, AA SEQ ID NO: SEQ 46), and CAIX-28ζ (a second-generation CAR structure containing a CD28 costimulatory domain) (DNA SEQ ID NO: SEQ 47, AA SEQ ID NO: SEQ 48). In this group of structures, all structures possess a FLAG tag, and all of the secreted BiTA antibodies have a His tag. The inclusion of the His tag facilitates subsequent detection of BiTA secretion levels. Details of these structures are shown in Figure 2.

1.3 Structural Design of tERBB2, CD3e-BBζ, HER2-BiTA, HER2-CAB, HER2-ζ, HER2-BBζ, and HER2-28ζ in the Experimental Group of Single-Chain Antibodies Targeting HER2 In the third experimental group, we tested the antitumor activity of the CAB platform using single-chain antibodies (scFv) targeting HER2. The single-chain antibody variable region sequences were derived from the antibody drug Herceptin (trastuzumab). In this example, we designed a third group of structures, including truncated ERBB2, CD3e-BBζ, HER2-CAB (DNA SEQ ID NO: SEQ 49, AA SEQ ID NO: SEQ 50), HER2-ζ (DNA SEQ ID NO: SEQ 51, AA SEQ ID NO: SEQ 52), HER2-BBζ (DNA SEQ ID NO: SEQ 53, AA SEQ ID NO: SEQ 54), and HER2-28ζ, a second-generation CAR construct (DNA SEQ ID NO: SEQ 55, AA SEQ ID NO: SEQ 56), for a total of seven structures. In this group of structures, all structures except HER2-ζ carry a FLAG tag, and all secreted BiTA antibodies also carry a His tag. Details are shown in Figure 3.

Example 2 Lentiviral Packaging of CAB and Its Control Construct In this disclosure, the inventors prepared CAB-T cells using lentivirus as a vector. First, the inventors prepared a lentiviral vector carrying a gene encoding CAB and its control construct. The specific procedure for packaging the lentivirus is as follows.

1) 1 × ¹⁰ HEK 293T cells were seeded in a 10 cm culture plate, and 10 mL of DMEM (Hyclone, SH30243.01) medium containing 10% FBS (Gibco, 10099-141C) was added, the cells were mixed thoroughly, and the cells were incubated at 37°C overnight.

2) The next day, when the cell density of HEK 293T (ATCC, CRL-3216) reached approximately 90%, the medium was replaced with serum-free DMEM.

3) Plasmid complexes were prepared. In this preparation, the amounts of various plasmids were 8 μg plasmid DNA, 4 μg psPAX2, and 2 μg pMD2, respectively. These were dissolved in 1 mL of opti-MEM (Gibco, 31985-070), 42 μL of PEI (Polysciences, 24765-2) was added, and the mixture was vortexed for 20 seconds. After leaving it at room temperature for 15 minutes, the mixture was gently added to HEK293T medium along the side, and the culture was maintained at 37°C.

4) After culturing the cells for 4 hours, the medium was removed, washed once with PBS (Hyclone, SH30256.01), and fresh pre-warmed DMEM medium containing 2% FBS was added again.

5) After 48 and 72 hours of transfection, the supernatants were collected and centrifuged at 2000 g for 5 minutes, and the precipitates were discarded. The supernatants were filtered through a 0.25 μm filter (Sartorius, 16541-K). PEG 8000 (Sigma, 89510-1KG-F) was then added to a final concentration of 5% and NaCl (Sigma, S5150-1L) to a final concentration of 0.15 M. The mixture was mixed vigorously and left overnight at 4°C.

6) The virus supernatant was centrifuged at 2000g and 4°C for 20 minutes, the supernatant was removed, and the virus pellet was dissolved in 50-100 μL of PBS and frozen at -80°C.

Example 3 Preparation of modified T cells carrying CAB and its control structure After completing the preparation of the lentiviral vector carrying the CAB structure, the lentiviral vector can be used to infect immune cells to complete the preparation of CAB-T cells. The specific procedure for preparing CAB-T cells is as follows.

1) Commercially available PBMC (Saily Bio, SLB-HP050B) cells were cultured at an initial cell density of 1 x 10 ⁶ /mL using X-VIVO 15 (LONZA, 04-418Q) containing 5% human blood albumin (GRIFOLS, 20% human blood albumin).

2) Anti-CD3/CD28 beads (Miltenyi Biotec, 130-091-441) were added at a cell:bead ratio of 3:1, and 1000 IU/mL IL-2 (Si Huan Sheng Wu, SFDA approval number: S10970016) was added to activate T cell expansion.

3) After 48 hours of cell activation, T cells were infected by adding an appropriate amount of virus and 12 μg/mL protamine (Sigma, P4005).

4) After 24 hours of lentiviral infection, the cell suspension was aspirated and complete fresh X-VIVO 15 medium was added at a concentration of 1 x ¹⁰⁶ cells/mL.

5) The cell density was monitored daily, and T cell culture solution containing 1000 IU/mL of IL-2 was added in a timely manner to maintain the T cell density at approximately 1 x ¹⁰ cells/mL, and the cells were allowed to continue expanding for 5-10 days to complete the preparation of CAR-T cells.

Example 4: Assay of CAB-T Cell Positive Frequency After the preparation of CAB-T and its control cells was completed, the infection efficiency was determined for subsequent activity analysis. Specifically, the method for detecting the CAB-T positive frequency using a FLAG antibody was as follows.

1) ^3-5x10 cells were harvested, 200µL of FACS buffer (PBS containing 1% FBS) was added to each flow cytometry tube, and the mixture was centrifuged at 300g for 5 minutes; biotin-CAIX (sino biological, 10107-H02H) was added to the CAIX-Truc sample at a final concentration of 100nM, and the mixture was incubated at 4°C for 20 minutes; biotin-HER-2 (ACRO, HE2-H82E2) was added to the Her2-Truc sample at a final concentration of 100nM, and the mixture was incubated at 4°C for 20 minutes; the supernatant was discarded, and 200µL of Fixation/Permeabilization solution (BD bioscience, 554715) was added, and the mixture was incubated at 4°C for 20 minutes.

2) The mixture was centrifuged at 300 g for 5 minutes; the supernatant was removed, 200 μL of 1x Perm/Wash buffer (BD bioscience, 554715) was added, the mixture was resuspended, and the mixture was centrifuged at 400 g for 5 minutes to wash twice.

3) The centrifuged supernatant was removed, and 100 μL of anti-Flag antibody diluted 1:1000 in FACS buffer was added to each sample. The cells were mixed thoroughly and incubated at 4°C for 30 minutes.

4) After incubation, 1 mL of FACS buffer was added to each flow cytometry tube, and the mixture was centrifuged at 400 g for 5 minutes.

5) The centrifuged supernatant was removed, 1 mL of FACS buffer was added, the mixture was resuspended, and centrifuged at 400 g for 5 minutes.

6) The centrifuged supernatant was removed, and 100 μL of SA-PE (Invitrogen, S866) diluted 1:250 with FACS buffer was added to each sample. The cells were mixed thoroughly and incubated in the dark at 4°C for 30 minutes. After incubation, 1 mL of FACS buffer was added to each flow cytometry tube, and the mixture was centrifuged at 300 g for 5 minutes. The centrifuged supernatant was removed, and the washing process was repeated twice.

7) The samples were run on a flow cytometer for detection.
result:
In the first experimental group, since the FLAG tag is carried by the CD3e-BBζ structure and the first CAIX-CAB structure, the FLAG antibody can be used to detect the positive frequency of T cells modified with the corresponding structure, while the biotin-labeled CAIX protein can be used to detect the positive frequency of T cell transduction for T cells modified with CAIX-TruC. However, since the first CAIX-BiTA structure does not have a suitable tag, the positive frequency of T cells modified with this structure cannot be detected. However, from the subsequent results, it can be determined that the positive frequency of T cells modified with the first CAIX-BiTA can meet the requirements of experimental analysis. The detection results are shown in Figure 4.

In the second and third experimental groups, we designed a FLAG tag for each construct, allowing the positive frequency of the corresponding modified T cells to be determined by labeling and detection with a FLAG antibody (for the first-generation HER2-ζ construct without a FLAG tag, biotinylated HER2 was used to detect transduction efficiency). NT represents non-transduced T cells and was used as a negative control group. The test results are shown in Figures 5 and 6. The difference in infection efficiency between different samples is within the acceptable limits.

From the above, it can be seen that the positive transduction frequencies for each group of modified T cells designed using the structures disclosed in the present disclosure and the structures disclosed in the prior art meet the requirements of experimental analysis.

Example 5 Antigen-Dependent Cytokine Release Assay of CAB-T When CAB-T cells are co-cultured with tumor cells, CAB-T can release multiple inflammatory cytokines by recognizing and activating target antigens on the surface of tumor cells. Based on this, in this example, the levels of cytokines released by activated CAB-T cells were detected by enzyme-linked immunosorbent assay (ELISA).

The ELISA detection procedure is as follows.
1) ^1x105 effector cells and ^1x105 target cells were seeded at 200 μL/well. The 96-well cell culture plate was co-cultured overnight and centrifuged at 300g for 5 minutes. 150 μL of supernatant/well was transferred to a new 96-well cell culture plate using a multichannel pipette, and cytokine detection was performed using IFN-γ (Invitrogen, 88-7316-88), IL-2 (Invitrogen, 88-7025-88), and TNF-α (Invitrogen, 88-7346-88) assay kits, respectively.

2) The ELISA plate was coated with human anti-IFN-γ/IL-2/TNF-α antibody one day in advance. The human anti-IFN-γ/IL-2/TNF-α antibody was diluted with PBS (1:250), and 100 μL of antibody was added to each well. The ELISA plate was sealed overnight at 4°C using a microplate sealer.

3) Plate washing: The liquid in the plate was quickly removed, and 200 μL of washing buffer was added per well using a multichannel pipette. This plate washing process was repeated five times.

4) Add 200 μL of 1x ELISA/ELISASPOT diluent to each well, cover with a microplate sealer, and shield at room temperature for 60 minutes.

5) Plate washing: The liquid in the plate was quickly removed, and 200 μL of washing buffer was added per well using a multichannel pipette. This plate washing process was repeated five times.

6) Human IFN-γ ELISA standards were prepared with eight gradients (in pg/mL): 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125.

7) Add 100 μL/well of the standards and samples to the ELISA plate. Dilute both the samples and standards to the desired concentrations with 1x ELISA/ELISASPOT diluent, cover with a microplate sealer, and incubate for 2 hours at room temperature.

8) Plate washing: The liquid in the plate was quickly removed, and 200 μL of washing buffer was added per well using a multichannel pipette. This plate washing process was repeated four times.

9) Human IFN-γ/IL-2/TNF-α detection antibody was diluted in PBS (1:250), and 100 μL of antibody was added to each well. The ELISA plate was sealed using a microplate sealer and incubated for 1 hour at room temperature.

10) Streptavidin-HRP conjugate was prepared by diluting (1:250) in PBS and added to the ELISA plate at 100 μL/well. The plate was covered with a microplate sealer and incubated for 30 minutes at room temperature.

11) Plate washing: The liquid in the plate was quickly removed, and 200 μL of washing buffer was added per well using a multichannel pipette. This plate washing process was repeated five times.

12) The TMB substrate was warmed to room temperature 30 minutes prior to incubation and added to the ELISA plate at 100 μL/well. After incubation at room temperature for 5-10 minutes, 50 μL/well of stop solution was added.

13) Absorbance was read using a microplate reader at a detection wavelength of OD = 450 nm.

14) A standard curve was calculated based on the concentration and OD value of the standard substance, and the concentration of the test sample was calculated based on the standard curve. The plot was created using GraphPad Prism mapping software.

result:
In the first experimental group, CAIX-CAB-T cells or their control group were cocultured with CAIX ⁺ HEK 293T cells or CAIX ^- HEK 293T cells, respectively, and the levels of inflammatory cytokines IL-2 and IFN-γ released in the supernatant were detected. The results are shown in Figure 7. When CAIX-CAB-T and its control group were cocultured with CAIX ^- HEK 293T cells, respectively, none of the immune cells showed significant cytokine release. When CAIX-CAB-T and its control group were cocultured with CAIX ⁺ HEK 293T cells, T cells modified with the first CAIX-BiTA, the first CAIX-CAB, and CAIX-TRuc all showed coculture time-dependent cytokine release levels. The cytokine levels accumulated after 48 hours of coculture were significantly higher than those accumulated after 24 hours of coculture. When unmodified T cells and CD3e-BBζ-modified T cells were cocultured with CAIX ⁺ HEK 293 T cells, respectively, no significant release of IL-2 or IFN-γ cytokines was detected. Surprisingly, the cytokine levels released by the first CAIX-CAB-T cells were found to be significantly higher than those of the first CAIX-BiTA-T cells after 48 hours of coculture. After mixing CD3e-BBζ-modified T cells and the first CAIX-BiTA-modified T cells at a 1:1 ratio and then cocultured with CAIX ⁺ HEK 293 T cells, the cytokine release levels were found to be comparable to those of the first CAIX-CAB-T cells. These results demonstrated that CAIX-CAB-T cell activation is dependent on the CAIX antigen and that BiTA and CD3e-BBζ synergistically promote T cell activation. We also demonstrated that CAB-T cells have comparable in vitro activation capacity to control TRuC-T cells.

In the second experimental group, CAIX-CAB-T and its control cells were co-cultured with CAIX ⁺ MB-231 or CAIX ^- MB-231 cells, respectively (CAIX expression levels are shown in Figure 8A), and the levels of released inflammatory cytokines IL-2, IFN-γ, and TNF-α in the supernatant were detected. The results are shown in Figure 8. When CAIX-CAB-T cells and their control were co-cultured with CAIX ^- MB-231 cells, respectively, neither cell showed significant cytokine release. After co-culture of CAIX-CAB-T and its control group with CAIX ⁺ MB-231 cells for 48 hours, T cells modified with CAIX-BiTA, CAIX-CAB, CAIX-BBζ, CAIX-28ζ, and CAIX-ζ exhibited various degrees of activation. The data show that T cells modified with CAIX-CAB-T and CAIX-BiTA, as well as first- and second-generation CARs, had similar IFN-γ and TNF-α release capacities, whereas CAIX-CAB-T released less IL-2 than second-generation CAR cells but slightly more IL-2 than CAIX-BiTA and first-generation CAR-modified T cells. The results of the second experimental group showed the dependence of CAIX-CAB-T cell activation on the CAIX antigen and differences in cytokine release compared to second-generation CARs, i.e., the IFN-γ and TNF-α release by CAIX-BiTA-T and CAIX-CAB-T was substantially equivalent to that by second-generation CARs, and CAIX-BiTA-T and CAIX-CAB-T were weaker than second-generation CARs in terms of IL-2 release.

Example 6: Antigen-Dependent Upregulation of T Cell Activation Markers in CAB-T When CAB-T cells are co-cultured with tumor cells, CAB-Ts can recognize and activate target antigens on the surface of tumor cells. The expression levels of T cell activation marker proteins on the membrane surface, including CD137, CD25, CD27, and the like, are significantly upregulated. The cell proliferation ability, indicated by the expression level of Ki67, is increased, and the killing ability of T cells, indicated by CD107a, is also enhanced. Based on this, in this example, changes in the expression levels of the above-mentioned membrane surface proteins in activated CAB-T cells were detected using the above-described staining method and flow cytometry.

The specific cell staining procedure was as follows:
1) ^1x105 effector cells and ^1x105 target cells were seeded at 200 μL/well in a 96-well cell culture plate. The cells were co-cultured overnight and centrifuged at 300 g for 5 minutes. 200 μL of FACS buffer was added to each well and centrifuged at 300 g for 5 minutes.

2) The centrifuged supernatant was removed, and the cells were resuspended by adding 200 μL of FACS buffer and centrifuged at 300 g for 5 minutes.

3) Antibodies were diluted in FACS buffer (100 μL/well):
BV421 mouse anti-human CD3 (BD Bioscience, 562426) 1:500 dilution PE mouse anti-human CD137 (BD Bioscience, 555956) 1:200 dilution APC mouse anti-human CD27 (BD Bioscience, 561786) 1:200 dilution PE-cy7 mouse anti-human CD25 (BD Bioscience, 557741) 1:200 dilution.

4) The centrifuged supernatant was removed, and 100 μL of antibody was added to each well, mixed, and incubated in the dark at 4°C for 30 minutes.

5) 200 μL of FACS buffer was added to each well, centrifuged at 300 g for 5 minutes, and the supernatant was removed.

6) The centrifuged supernatant was removed and step 2.5 was repeated.
7) The supernatant was discarded, and 200 μL of Fixation/Permeabilization solution (BD bioscience, 554715) was added, followed by incubation at 4° C. for 20 minutes.

8) Centrifugation was performed at 300 g for 5 minutes. The supernatant was removed, and 200 μL of 1x Perm/Wash buffer (BD bioscience, 554715) was added for resuspension. Centrifugation was performed at 400 g for 5 minutes.

9) Wash twice and then dilute the antibody FITC mouse anti-Flag (Biolegend, 637318) at a 1:1000 dilution in FACS buffer.

10) Centrifugation was performed at 400 g for 5 minutes. The supernatant was removed and the mixture was washed twice.

11) Flow cytometry detection was performed using FSC/SSC gating to obtain the desired lymphocyte population (PBMC), and CD3 BV421 ⁺ and Flag FITC ⁺ cell populations were selected to obtain live CAR-T cells, which were then gated using non-virally transduced PBMC as a standard to obtain the percentage of CAR-T cells CD137 PE ⁺ cells.

result:
In the first experimental group, CAIX-CAB-T cells or their control group cells were co-cultured with CAIX ⁺ HEK 293T cells or CAIX ^- HEK293T cells, respectively, to detect changes in the expression levels of CD137 and CD107a on the T cell membrane surface. The results are shown in Figure 9. When CAIX-CAB-T and its control group were co-cultured with CAIX ^- HEK 293T cells, the expression levels of CD137 and CD107a in CAB-T and its control group immune cells did not change significantly. After coculture of CAIX-CAB-T and its control group with CAIX ⁺ HEK 293T cells for 24 hours, the expression levels of CD137 and CD107a in T cells modified with the first CAIX-BiTA, the first CAIX-CAB, and CAIX-TRuC were significantly upregulated, with the first CAIX-CAB-T showing a much higher upregulation level than the first CAIX-BiTA. When unmodified T cells and T cells modified with CD3e-BBζ were cocultured with CAIX ⁺ HEK 293T cells, no significant upregulation of CD137 or CD107a was detected. When CD3e-BBζ-modified T cells and the first CAIX-BiTA-modified T cells were mixed at a 1:1 ratio and then cocultured with CAIX ⁺ HEK 293T cells for 24 hours, the expression levels of CD137 and CD107a were found to be significantly higher than those of CD3e-BBζ-T and the first CAIX-BiTA-T cells cocultured individually with CAIX ⁺ HEK 293T cells. These results demonstrate that CAIX-CAB-T cell activation is dependent on the CAIX antigen and that BiTA and CD3e-BBζ synergistically promote T cell activation. CAB-T cells also demonstrated comparable in vitro activation capacity to control TRuC-T cells.

In the second experimental group, CAIX-CAB-T cells and their control group cells were co-cultured with CAIX ⁺ MB-231 cells or CAIX ^- MB-231 cells for 48 hours, respectively, and then changes in the expression levels of CD137, CD25, CD27, and Ki67 on the T cell membrane surface were detected. The results are shown in Figure 10. When CAIX-CAB-T and its control group were co-cultured with CAIX ^- MB-231 cells, respectively, the expression levels of CD137, CD25, CD27, and Ki67 in CAIX-CAB-T and its control group immune cells did not change significantly. After coculture of CAIX-CAB-T and its control group with CAIX ⁺ MB-231 cells, the expression levels of CD137, CD25, CD27, and Ki67 in T cells modified with CAIX-BiTA, CAIX-CAB, first-generation CAR, and second-generation CAR were significantly upregulated. When T cells modified with tERBB2 and CD3e-BBζ were cocultured with CAIX ⁺ MB-231 cells, respectively, no significant upregulation of CD137, CD25, CD27, or Ki67 was detected. These results indicate that CAIX-CAB-T cell activation is dependent on the CAIX antigen and also demonstrate that CAB-T cells have in vitro activation capabilities comparable to those of control BiTA-T, first-generation CAR, and second-generation CAR cells.

In the third experimental group, we examined the in vitro activation ability of a HER2-CAB construct constructed using a trastuzumab-derived scFv sequence. The results are shown in Figure 11. HER2-CAB-T cells and their control cells were co-cultured with the HER2-positive SKBR3 cell line or the HER2-negative Raji cell line for 48 hours, and then changes in the expression levels of CD137, CD25, CD27, and Ki67 on the T cell membrane surface were detected. The results are shown in Figure 12. When HER2-CAB-T and its control cells were co-cultured with Raji cells, the expression levels of CD137, CD25, CD27, and Ki67 in HER2-CAB-T and its control immune cells did not change significantly. After co-culture of HER2-CAB-T and its control group with HER2-positive SKBR3 cells, the expression levels of CD137, CD25, CD27, and Ki67 in T cells modified with HER2-CAB, first-generation CAR, and second-generation CAR were significantly upregulated. When T cells modified with tERBB2 and CD3e-BBζ were co-cultured with SKBR3 cells, respectively, no significant upregulation of CD137, CD25, CD27, or Ki67 was detected. These results indicate that HER2-CAB-T cell activation is dependent on the HER2 antigen and also demonstrate that CAB-T cells have in vitro activation capabilities equivalent to those of the control group BiTA-T, first-generation CAR, and second-generation CAR cells.

Example 7 Analysis of Unmodified T Cells Activated by Paracrine CAB-T The initial intention behind the design of the CAB structure was to achieve the following effect: upon encountering tumor cells, CAB-T would activate its own antitumor activity, while unmodified T cells surrounding the CAB-T would be activated by the BiTA drug secreted by the CAB-T to perform paracrine activation. To detect the paracrine activation function of CAB-T in T cells, the inventors performed a Transwell experiment. Specifically, the experiment was conducted by separating CAB-T and unmodified T cells using a physical barrier with a 0.4 μm Transwell system, with CAB-T cells placed in the upper chamber and unmodified T cells and tumor cells placed in the lower chamber. The soluble BiTA secreted by CAB-T could freely enter the lower chamber through the 0.4 μm grid and activate the ability of the unmodified T cells below to activate by recognizing tumor cells.

The specific experimental procedure was as follows:
1) ^1x106 BiTA-T cells and ^1x106 CAB-T cells were resuspended in 200µL of X-VIVO 15 medium, respectively, and then added to the upper chamber of a 0.4µm Transwell (Coning, 3413).

2) 1×10 ⁶ unmodified T cells and 1×10 ⁶ target cells were resuspended in 500 μL of X-VIVO-15 medium and added to the lower chamber of the Transwell.

3) The upper chamber was carefully placed into the lower chamber and incubated in an incubator for 48 hours.

4) The culture plate after overnight co-culture was removed, and 500 μL of cell supernatant in the lower chamber was removed. After centrifugation at 300 g for 5 minutes, 150 μL of supernatant per well was removed using a multichannel pipette and transferred to a new 96-well cell culture plate for ELISA detection of IFN-γ/IL-2/TNF-α.

result:
In the second experimental group (Figure 12), both BiTA secreted by CAIX-BiTA-T and BiTA secreted by CAIX-CAB-T were able to activate the ability of unmodified T cells underlying the cells to recognize CAIX-positive MB-231 tumor cells per Transwell pore, and showed high levels of IFN-γ and TNF-α release. In addition, we observed no significant change in IL-2 release levels, which was consistent with the results of the study in Example 9.

The results shown in the third experimental group (Figure 13) indicate that HER2-CAB-T also exhibited the same paracrine activation of the ability of unmodified T cells to recognize tumor antigens. HER2-CAB-T cells were able to activate unmodified T cells in the Transwell below to recognize HER2-positive SKBR3 tumor cells, whereas control tERBB2-T cells were not. HER2-CAB-T did not aid in the recognition of HER2-negative Raji cells by unmodified T cells.

Both the second and third experimental groups demonstrated that the CAB-T construct was able to activate the ability of unmodified T cells to recognize tumor cells via paracrine BiTA.

Example 8 Analysis of Immune Checkpoint Expression Levels and Cellular Differentiation Phenotypes of CAB-T Cells The expression levels of immune checkpoint proteins on immune cells and the differentiation phenotype of immune cells are significantly related to the therapeutic efficacy of adoptive T cells. Therefore, both lower expression levels of immune checkpoints and a higher proportion of memory T cells predict better clinical response rates. The inventors used flow cytometry to detect the effects of CAB structures on the immune checkpoint expression levels and cellular phenotypes of T cells modified with the CAB structure.

The specific analytical procedures were as follows:
1) The 96-well cell culture plate that had been co-cultured overnight was centrifuged at 300 g for 5 minutes, and 200 μL of FACS buffer was added to each well, followed by centrifugation at 300 g for 5 minutes.

2) The supernatant was removed, and the cells were resuspended in 200 μL of FACS buffer and centrifuged at 300 g for 5 minutes.

3) Antibodies were diluted with FACS buffer to prepare the antibody mix (100 μL/well).

4) The centrifuged supernatant was removed, and 100 μL of the antibody mix was added to each well and incubated in the dark at 4°C for 30 minutes.

5) 200 μL of FACS buffer was added to each well, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.

6) The centrifuged supernatant was removed and step 2.5 was repeated.
7) 200 μL of Fixation/Permeabilization solution (BD bioscience, 554715) was added, and the mixture was incubated at 4° C. for 20 minutes.

8) Centrifugation was performed at 300 g for 5 minutes; the supernatant was removed, and 200 μL of 1x Perm/Wash buffer (BD bioscience, 554715) was added, followed by resuspension and centrifugation at 400 g for 5 minutes; washing was performed twice.

9) Antibody FITC mouse anti-Flag (Biolegend, 637318) was diluted 1:1000 in FACS buffer.

10) Centrifuge at 400g for 5 minutes; remove the supernatant and wash twice.

11) Flow cytometry detection was performed using FSC/SSC gating to obtain the desired lymphocyte population, and CD3 BV421 ⁺ and Flag FITC ⁺ cell populations were selected to obtain live CAR-T cells.

result:
The results of the second experimental group ( FIG. 14 ) show that, after coculture with CAIX-positive MB-231 cells, the expression levels of immune checkpoint proteins, including LAG-3, PD-1, and TIM-3, on the surface of CAIX-CAB-T cells were lower than those of second-generation CAR-T modified with CAIX-28ζ, the expression levels of PD-1 and TIM-3 on CAB-T cells were essentially the same as those of CAIX-BBζ second-generation CAR-T, and the expression level of LAG-3 in CAB-T cells was slightly lower than that of second-generation CAR-T modified with CAIX-BBζ. The lower expression levels of immune checkpoint proteins indicated the clinical superiority of CAB-T cells compared with second-generation CAR-T.

In addition, in the second experimental group, we also used CD45RA and CCR7 to analyze the differentiation phenotype of immune cells. The differentiation marker proteins were primary T cells (CD45RA ⁺ , CCR7 ⁺ ), central memory T cells (CD45RA ⁻ , CCR7 ⁺ ), effector memory T cells (CD45RA ⁻ , CCR7 ⁻ ), and terminally differentiated effector T cells (CD45RA ⁺ , CCR7 ⁻ ), respectively. Compared with second-generation CAR-T, the proportions of cells with the primary differentiation T cell phenotype and central memory T cell phenotype of CAIX-CAB-T were significantly higher than those of second-generation CAR-T cells, while the proportion of effector memory T cells of second-generation CAR-T cells was significantly higher than that of CAR-T cells, as can be seen from the results shown in Figure 14D. A more abundant central memory T cell phenotype predicted better clinical efficacy, thus demonstrating the superiority of CAB-T in differentiated state compared to second-generation CAR-T.

The results shown in the third experimental group (Figure 15) indicate that the immune checkpoint expression status and cell phenotype differentiation results for HER2-CAB-T were essentially consistent with the analysis results for CAIX-CAB-T. That is, the immune checkpoint expression levels of HER2-CAB-T were lower than those of second-generation CAR-T modified with CAIX-28ζ, and were essentially equal to or slightly lower than those of second-generation CAR-T modified with CAIX-BBζ. The differentiation status of HER2-CAB-T also had a higher proportion of central memory T cell phenotype than second-generation CAR-T.

Example 9 Antigen-Dependent Killing Activity of CAB-T Whether CAB-T cells have in vitro killing activity is an important criterion for determining the potential clinical efficacy of CAB-T. To verify the tumoricidal activity of CAB-T, the present inventors used the LDH method for detection.

The specific experimental procedure was as follows:
1) Experimental wells, effector cell control wells, target cell control wells, target cell maximum release wells, medium control wells, and volume control wells were arranged respectively; the experimental procedure was carried out according to the standard procedure of CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, G1781).

2) Different target-effector ratios, i.e., the number of effector cells: the number of target cells, were set at 0:1, 1:1, 5:1, 10:1, and 20:1.

3) Number of cells: 1 x ¹⁰⁴ target cells, 50 μL/well.
4) For the experimental wells, 100 μl of cells (50 μL effector cells + 50 μL target cells) at different dilution ratios of effector cells:target cells = 0:1, 1:1, 5:1, 10:1, and 20:1 were added to the cell culture plate in triplicate.

5) Effector cell control wells contained effector cells:target cells at ratios of 0:0, 1:0, 5:0, 10:0, and 20:0 in duplicate.

6) Target cell control wells received 50 μL of target cells at 1×10 ⁴ /well and 50 μL of medium.

7) To the target cell maximum release wells, 50 μL of target cells at 1×10 ⁴ and 50 μL of medium were added, and 10 μL of lysate was added 1 hour before collecting the samples.

8) To the medium control wells, 100 μL of medium was added.
9) 100 μL of medium was added to the volume control wells, and 10 μL of lysate was added to the target cell maximum release wells 1 hour before sample collection, but in the meantime 10 μL of lysate was added and incubation was carried out at 37°C.

10) According to the designed layout, the samples were added to the plate and incubated at 37°C, 5% _CO2 for 24 hours, 36 hours, or 48 hours.

11) Remove the assay buffer from the -20°C refrigerator and thaw it in the dark at 4°C. Before use, add 12 ml of assay buffer to the flask containing the substrate mix and mix.

12) The culture plate was centrifuged at 250 g for 4 minutes, and 50 μL/well of the cell supernatant was transferred to a new ELISA plate.

13) 50 μL/well of substrate mix was added to a new ELISA plate (12 mL of assay buffer was added to the mix flask and mixed in the dark).

14) Incubate at room temperature for 30 minutes in the dark, then add 50 μL/well of stop solution.

15) Absorbance was read using a microplate reader at a detection wavelength of OD = 490 nm for 1 hour.

16) Cell mortality (%) was calculated based on the OD value.
Experimental well = effector-target ratio - medium control (average)
Spontaneous release of target cells = target cell control - medium control (mean)
Spontaneous release of effector cells = effector cell control - medium control (mean)
Maximum release of target cells = Maximum release of target cells (mean) - Volume control (mean)
Cell lethality (%) = (experimental - spontaneous release of target cells - spontaneous release of effector cells) / (maximum release of target cells - spontaneous release of target cells).

result:
BiTAs secreted by CAB-Ts can activate the CAB-T cells themselves and their surrounding non-modified T cells in a target antigen-dependent manner, leading to the killing of target antigen-positive tumor cells; on the other hand, CAB-T cells express CD3e-BBζ, and thus CAB-Ts can depend on the activation of endogenous TCRs. Furthermore, CD3e-BBζ can increase the activation level of CAB-T cells, thereby promoting CAB-Ts to release more BiTAs; these effects mutually reinforce each other. Therefore, in theory, CAB-Ts should have a stronger lethal effect on tumor cells than BiTA-Ts.

In the first experimental group, to detect the lethal effect of CAIX-CAB-T cells on CAIX ⁺ HEK 293T target cells, we selected CAIX-CAB-T cells as effector cells in the experimental group for detecting the lethal effect. The effector cells were co-cultured with target cells at effector-to-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1 for 24 and 48 hours, respectively, and the supernatants were collected to determine the ability of T cells to kill target cells at different effector-to-target ratios. The results in Figure 16 show that the first CAIX-CAB-T cells and their control T cells had no lethal effect on CAIX ^- HEK293T cells, whereas the first BiTA-T cells, the first CAIX-CAB-T, CAIX-TRuC-T, and the mixture of CD3e-BBζ-T and the first BiTA-T cells exhibited varying degrees of lethality against CAIX ⁺ HEK293T cells, and this lethality increased with increasing effector-to-target ratio. Furthermore, the killing of target cells by CAIX-CAB-T and its control T cells became more significant with increasing coculture time, with the lethal effect of 48 hours of coculture being significantly stronger than that of 24 hours of coculture. CD3e-BBζ-T and the unmodified T cell control group did not exhibit a lethal effect against CAIX ⁺ HEK 293T cells. In addition, the first CAIX-CAB-T, CAIX-TRuC-T, and the mixed T cells of CD3e-BBζ-T and the first BiTA-T had comparable lethal abilities against CAIX ⁺ HEK 293T cells, and their lethal abilities were superior to those of the first BiTA-T. From these results, the inventors can conclude that the lethal ability of CAIX-CAB-T against tumor cells depends on the expression of its target antigen and that its lethal ability was comparable to that of the control CAIX-TRuC-T cells. Furthermore, BiTA-T and CD3e-BBζ-T also exhibited a synergistic effect on target cell killing.

The method for the second experimental group was identical to that for the first experimental group. We detected the killing ability of CAIX-CAB-T and its control cells against CAIX ⁺ MB-231 or CAIX ⁻ MB-231 tumor cells. Effector cells were cocultured with target cells for 36 hours at effector-to-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1, respectively, and supernatants were collected to determine the ability of T cells to kill target cells at different effector-to-target ratios. The results shown in Figure 17 indicate that CAIX-CAB-T and its control cells had no lethal effect on CAIX ⁻ MB-231 control tumor cells, and that CAIX-CAB-T and first- and second-generation CAR-T cells targeting CAIX showed comparable killing ability against CAIX ⁺ MB-231 cells. In addition, tERBB2-T and CD3e-BBζ-T control cells lacked the ability to kill CAIX ⁺ MB-231 cells. CAIX-CAB-T was found to have comparable lethal ability to first- and second-generation CAR-T against tumor cells, and this killing ability was target antigen-dependent. Note that CAIX-CAB-T and CAIX-BiTA-T did not show differences in their lethal ability against target cells in this experimental group due to higher transduction levels or different donor cell sources or the like.

The third experimental group was identical to the first and second experimental groups. We examined the ability of HER2-CAB-T and its control cells to kill the HER2-positive tumor cells SKBR3 or the HER2-negative tumor cells Raji. Effector cells were co-cultured with target cells for 36 hours at effector-to-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1, respectively, and supernatants were collected to determine the ability of T cells to kill target cells at different effector-to-target ratios. The results shown in Figure 18 demonstrate that HER2-CAB-T and its control cells had no lethal effect on HER2-negative Raji cells, and that HER2-CAB-T and first- and second-generation CAR-T cells exhibited comparable lethal ability against SKBR3. In addition, tERBB2-T and CD3e-BBζ-T control cells lacked the ability to kill SKBR3 cells. HER2-CAB-T was found to have the same lethal ability against tumor cells as first- and second-generation CAR-T, and this lethal ability was target antigen-dependent.

Example 10 Comparison of In Vitro Activity of Different CAB Structures with BiTA and Second-Generation CAR Because the expression levels of CD3e-BBζ and BiTA in cells differ, the present inventors attempted to analyze the differences between different structures of CAB, BiTA, and second-generation CAR. The present inventors first designed a group of CABs and their control structures, including tERBB2, HER2-BiTA, HER2-CAB, HER2-CAB ^R (DNA SEQ ID NO: SEQ 67, AA SEQ ID NO: SEQ 68), and HER2-BBζ, as shown in FIG. 19 . Using vectors with the above structures, lentiviruses were packaged according to the method described in Example 2 and infected into T cells according to the method described in Example 3. The positive frequency of infected cells was detected according to the method described in Example 4, and the detection results are shown in FIG. 20 . The positive frequencies of modified T cells were essentially equal.

To identify the in vitro activation ability of the modified T cells, the modified T cells were co-cultured with HER2-negative Raji cells and HER2-positive SKBR3 cells, respectively, and then the released levels of cytokines IL-2 and IFNγ were detected according to the method described in Example 5. The results are shown in Figure 21. The cytokine release levels after activation of HER2-CAB-T and HER2-CAB ^R -T cells were essentially equal.

To identify differences in the in vitro killing ability of the modified T cells, we detected the in vitro killing ability of each of the modified T cells against Raji and SKBR3 cells according to the method described in Example 9. Compared with the HER2-CAB structure, the HER2-CAB ^R structure has a BiTA in front and a CD3e-BBζ structure in back, so theoretically, HER2-CAB ^R -T can secrete higher levels of BiTA. We speculated that HER2-CAB ^R -T would have better in vitro killing ability than HER2-CAB-T cells. As shown in Figure 22, the results were consistent with our hypothesis, i.e., HER2-CAB ^R -T showed superior in vitro killing ability to HER2-CAB-T cells.

Example 11 In Vivo Efficacy of CAIX CAB-T The antitumor activity of CAB-T in a mouse tumor model is an important criterion for determining the potential clinical efficacy of CAB-T. To verify the antitumor activity of CAB-T in a mouse tumor model, the following validation experiment was carried out.

The specific experimental procedure was as follows.
CAIX ⁺ MDA-MB-231 cell expansion and seeding 1) Sufficient CAIX ⁺ MDA-MB-231 cells were cultured and expanded in vitro, and the cells were harvested after trypsin digestion, washed three times with PBS, counted, and adjusted to a cell density of 15 x ¹⁰⁶ cells/ml using 80% RPMI-1640 basal medium containing 20% Matrigel. The cells were placed in a 50ml centrifuge tube, the opening of the centrifuge tube was covered with a sealing film and sealed, and the centrifuge tube was transferred to an SPF-grade animal room via a transfer window.

2) 68 female 8-week-old NCG severely immunodeficient mice purchased in advance were fed an adapted diet for one week, and then the hair on the right flank of each mouse was removed with a razor. CAIX ⁺ MDA-MB-231 cells at a density of 15 × 10 ⁶ cells/ml were dissociated and thoroughly mixed with a 1-ml pipette. 0.2 ml of the cells were subcutaneously inoculated into the right flank of each NCG mouse with a 1-ml syringe. That is, each NCG mouse was inoculated with 3 × 10 ⁶ CAIX ⁺ MDA-MB-231 cells. The cells were observed daily for subcutaneous tumor formation in the NCG mice, and each NCG mouse was numbered using a numbered ear tag 6 days after inoculation.

Grouping, administration, and measurement of CAIX ⁺ MDA-MB-231 tumor-bearing mice 3) Ten days after inoculation, the widest axis W and longest axis L of the subcutaneous tumor in the right flank of each NCG mouse were measured using a vernier caliper, and each mouse was weighed using an electrobalance. The subcutaneous tumor volume in the right flank of each NCG mouse was calculated according to tumor volume T = 1/2 × W × W × L. Mice with tumors that were too large or too small were excluded, and the NCG mice were evenly divided into 11 groups of 6 mice per group according to the average tumor volume.

4) Each group was divided into groups according to the following administration scheme and injected with the corresponding reagent or cells. In the hCAIX-BiTA (DNA SEQ ID NO: SEQ 71, AA SEQ ID NO: SEQ 72), hCAIX-CAB (DNA SEQ ID NO: SEQ 73, AA SEQ ID NO: SEQ 74), and hCAIX-BBζ (DNA SEQ ID NO: SEQ 75, AA SEQ ID NO: SEQ 76) structures for modifying T cells, the antibody sequences for recognizing CAIX are all derived from the humanized VHH amino acid sequence of SEQ ID NO: 18.

Dosing scheme for each group

5) The tumor volume and body weight of the mice were measured twice a week. For the final 37 days after tumor cell inoculation, the body weight and tumor volume of the mice were measured. After the mice were euthanized, the tumors of each mouse were excised, weighed, and photographed.

Results and Discussion The tumor growth curves, tumor photographs, and tumor weights for each group of mice are shown in the figures. The tumors in the PBS and NT groups rapidly increased with the extension of the inoculation time. This indicates that the CDX transplantation model was successfully established in this experiment. Compared with the PBS and NT groups, the hCAIX-BiTA-T cell and hCAIX-BBζ-T cell groups showed an inhibitory effect on tumor growth only at 2.5 × ¹⁰ /mouse, whereas the hCAIX-CAB-T cell group showed some effect at all three doses. These effects were dose-dependent; all tumors regressed in the 2.5 × ¹⁰ /mouse group, and five tumors regressed in the 0.75 × ¹⁰ /mouse group. The hCAIX-CAB-T cell group was significantly superior to the hCAIX-BiTA-T cell and hCAIX-BBζ-T cell groups at the same doses. The tumor growth curves, tumor photographs, and tumor weights of each group were consistent (Figures 23A, C, and D). There was no significant increase in body weight in any group throughout the experimental period (Figure 23B). This indicates the safety of each test sample.

Example 12 In Vivo Efficacy of HER2 CAB-T The antitumor activity of HER2 CAB-T was verified in the M-NSG immunodeficient mouse tumor model by the following experiment.

NCI-N87 cell expansion and seeding 1) NCI-N87 cells were cultured and expanded in vitro, and the cells were harvested after trypsin digestion, washed three times with PBS, counted, and adjusted to a cell density of 10 x ¹⁰ cells/ml using 80% RPMI-1640 basal medium containing 20% Matrigel. The cells were placed in a 50 ml centrifuge tube, the opening of the centrifuge tube was covered with a sealing film and sealed, and the centrifuge tube was transferred to an SPF-grade animal room via a transfer window.

2) Thirty-two female 8-week-old NSG severely immunodeficient mice were fed an adaptive diet, and the hair on the right flank of each mouse was removed with a razor. NCI-N87 cells at a density of 10 x ¹⁰ cells/ml were dissociated and thoroughly mixed with a 1 ml pipette. 0.2 ml of the cells were subcutaneously inoculated into the right flank of each NSG mouse with a 1 ml syringe. That is, each NSG mouse was inoculated with 3 x ¹⁰ NCI-N87 cells. The cells were observed daily for subcutaneous tumor formation in the NSG mice, and each NSG mouse was numbered using a numbered ear tag 6 days after inoculation.

Grouping, administration, and measurement of NCI-N87 tumor-bearing mice 3) Six days after inoculation, the widest axis W and longest axis L of the subcutaneous tumor in the right flank of each NSG mouse were measured using a vernier caliper, and each mouse was weighed using an electrobalance. The subcutaneous tumor volume in the right flank of each NCG mouse was calculated according to tumor volume T = 1/2 × W × W × L. Mice with tumors that were too large or too small were excluded, and the NSG mice were evenly divided into four groups of six mice per group according to the average tumor volume.

4) Each group was divided into groups and injected with the corresponding reagent or cells according to the following administration scheme: NT (DNA SEQ ID NO: SEQ 33, AA SEQ ID NO: SEQ 34), HER2 CAB ^R -T (DNA SEQ ID NO: SEQ 67, AA SEQ ID NO: SEQ 68), HER2 CAB-T (DNA SEQ ID NO: SEQ 49, AA SEQ ID NO: SEQ 50), and HER2 CAR-T structures for modifying T cells, all of which have scFv antibody sequences for recognizing HER2 derived from the trastuzumab amino acid sequence, SEQ ID NO: 66.

Dosing scheme for each group

5) The tumor volume and body weight of the mice were measured twice a week. For the final 48 days after tumor cell inoculation, the weight and tumor volume of the mice were measured. After the mice were euthanized, the tumors of each mouse were excised, weighed, and photographed.

Results and Discussion Tumor growth curves and tumor photographs for each group of mice are shown in the figures. Tumors in the NT group mice rapidly increased with the extension of the inoculation time. This indicates that the CDX transplantation model was successfully established in this experiment. Compared to the NT group, all three other modified T cell treatment groups showed certain tumor growth inhibition. In contrast to the mild tumor growth inhibition and two tumor-free mice in the HER2 CAR-T treatment group, both HER2-CAB ^R -T and HER2-CAB-T showed much better tumor growth inhibition, with five tumor-free mice and two tumor-free mice, respectively.

The tumor growth curves, tumor photographs, and tumor weight results for each group were consistent (Figures 24A, C, and D). There was no significant increase in the body weight of mice in each group throughout the experimental period. This indicates that each test sample had good safety profiles.

Example 13: Dasatinib acts as a safety switch to inhibit cytokine release in CAB-T. In the case of live cell therapy, CAB-T can lead to cytokine release syndrome (CRS) and on-target and off-tumor toxicities. Clinical management of CRS involves the anti-IL-6R agonist tocilizumab and steroid treatment. Here, the present inventors developed a method to manage potential toxicities using dasatinib.

Dasatinib was developed as an inhibitor of the BCR-ABL fusion protein and has been approved for clinical use in the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia. Additionally, it has been reported that dasatinib can block lymphocyte-specific protein tyrosine kinase (LCK), thereby disrupting CAR and TCR signaling by inhibiting the phosphorylation of CD3ζ and ZAP70. The following example demonstrates that dasatinib can dramatically inhibit cytokine release from CAB-T.

1 × ¹⁰ effector cells (CAIX CAB-T or HER2 CAB-T) and 1 × ¹⁰ target cells (CAIX ⁺ MDA-MB231 or SKBR3) were seeded into a 96-well cell culture plate at 200 μL/well with gradient concentrations of dasatinib (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 0 nM). After overnight co-culture, the plate was centrifuged at 300 g for 5 minutes. 150 μL of supernatant per well was then transferred to a new 96-well cell culture plate using a multichannel pipette. Cytokine detection was performed as described in Example 5.

As shown in Figures 25A and 25B and 26A and 26B, dasatinib effectively inhibited IFNγ and IL-2 secretion in both activated CAIX CAB-T and HER2 CAB-T at a low dose of approximately 25 nM. This result suggests that dasatinib could be used as a safety switch to control potential CRS syndrome and on-target, off-tumor toxicity of CAB-T in clinical treatment.

Example 14 Dasatinib Acts as a Safety Switch and Inhibits the Lethal Activity of CAB-T As described in Example 13, T cell therapy has the potential problem of on-target, off-tumor toxicity in clinical settings. To manage the potential toxicity of CAB-T, dasatinib was used to evaluate inhibition of the lethal activity of CAB-T.

We used the LDH cytotoxicity assay method to detect the lethality-inhibiting ability of dasatinib. The detailed protocol is described in Example 9. Briefly, 5 × ¹⁰ effector cells (CAIX CAB-T or HER2 CAB-T) and 1 × ¹⁰ target cells (CAIX ⁺ MDA-MB231 or SKBR3) were cocultured with gradient concentrations of dasatinib (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 0 nM), and the cytotoxicity of CAB-T against the target cells was determined at 2-hour intervals over a 12-hour period [effector to target cell (E:T) ratio, 5:1].

As shown in Figures 25C and 26C, dasatinib effectively inhibited the cytotoxicity of CAIX CAB-T and HER2 CAB-T against their target cells at a low dose of approximately 50 nM. This result suggests that dasatinib could be used as a safety switch to control potential on-target, off-tumor toxicity of CAB-T in clinical treatment.

Summary and Discussion:
The CAB-T technology utilizes BiTAs secreted by the CAB-T cells to simultaneously recognize chimeric CD3 or tumor antigens and endogenous CD3 in T cells, subsequently inducing tumor antigen-dependent activation of endogenous TCRs and chimeric CD3. Both activation of the endogenous TCR complex and activation of chimeric CD3 depend on the expression and secretion levels of BiTAs by CAB-T cells. Therefore, BiTAs can induce CAB-T cells via autocrine mechanisms and non-modified T cell activation in the tumor microenvironment via paracrine mechanisms. Meanwhile, CAB-T cells can release high levels of BiTAs into tumor tissues after activation in the tumor tissue, thereby mobilizing more non-modified T cells and promoting anti-tumor effects.

Therefore, compared to TRuC-T and TAC-T, CAB-T not only has the advantage of activating endogenous TCR signals, but also mobilizes the anti-tumor activity of infiltrating T cells into tumor tissue, theoretically offering greater therapeutic potential for solid tumors. In addition, unlike BiTE drugs, BiTA drugs are continuously secreted by CAB-T cells, solving the clinical application problem of the short half-life of single BiTE drugs. Furthermore, BiTAs targeting target antigens exert their maximum effect in the tumor microenvironment where CAB-T cells reach, rather than becoming highly concentrated in non-tumor tissue sites. Therefore, BiTAs have better safety and greater clinical potential compared to systemic administration of single BiTE drugs.

The mechanism of action of CAB-T and its potential clinical applications are described below.
1) In tumor tissues, BiTAs underexpressed in CAB-T cells can bind to their own endogenous TCR complexes or CD3e-BBζ in an autocrine manner (Figure 27B ), thereby stimulating CAB-Ts to release more BiTAs and establishing a local activation loop (Figure 27C ). This allows CAB-Ts to reach their maximum activation level at the local tumor site, thus exerting their maximum antitumor effect and achieving safety and efficacy similar to that of local drug administration to the tumor site. CD3e-BBζ was predicted to form heterodimers with endogenous CDδ or CD3γ chains on the T cell membrane, thus further enhancing CD3-based signaling.

2) Binding and activation of chimeric CD3e in CAB-Ts by autocrine BiTA (Figure 27A ) confers enhanced proliferation and activation capabilities to CAB-Ts compared to unmodified T cells, thus further enhancing the antitumor activity of CAB-Ts.

3) The autocrine and paracrine activation of CAB-T (Figures 27A , B, and C) and unmodified T cells (Figure 27D ) by BiTAs, respectively, solves the problem of CAR-T cells being unable to activate endogenous TCR signals, thus providing the possibility of treating solid tumors with CAB-T cells.

4) CAB-T can be used as a drug synthesis factory for BiTA, which solves the problem of BiTA's in vivo half-life. CAB-T activation depends on the release level of BiTA, and these two are interdependent and cooperate with each other to jointly determine the safety and efficacy of CAB-T for clinical application.

A schematic diagram of the mechanism of action of CAB-T is shown in FIG.
All documents mentioned in this disclosure are incorporated by reference herein as if each document were individually incorporated by reference. In addition, it should be understood that those skilled in the art, upon reading the above teachings of the present disclosure, will be able to make various modifications or variations of the present disclosure, and that equivalents thereof are also within the scope defined by the claims appended hereto.

Claims (25)

1. A nucleic acid molecule encoding a chimeric CD3 fusion protein and a bispecific T cell activation element, comprising:
the chimeric CD3 fusion protein comprises one or more polypeptides capable of being recognized by an anti-CD3 antibody (EC) , a transmembrane domain (TM), and a CD3 signal activation domain , wherein EC is a polypeptide from or derived from CD3e;
1. A nucleic acid molecule, wherein the bispecific T cell activating element is a fusion protein comprising one or more tumor antigen recognition regions and one or more CD3 antigen-binding antibody fragments. 2. The nucleic acid molecule of claim 1, wherein the chimeric CD3 fusion protein comprises one or more of the following domains: a hinge or linker region, and a costimulatory domain. The fusion protein has the structure of Formula I:
L-EC-H-TM-C-CD3ζ(I)
(In the formula,
L is absent or is a signal peptide sequence;
EC is a polypeptide binding domain that can be recognized by an anti-CD3 antibody and binds to said anti-CD3 antibody;
H is absent or is a linker or hinge region;
TM is the transmembrane domain;
C is absent or a costimulatory signaling molecule;
CD3ζ is a cytoplasmic signaling sequence that is absent or derived from CD3ζ;
Each "-" is independently a linker peptide or a peptide bond.
The nucleic acid molecule of claim 1 , The bispecific T cell activation element has the structure shown in Formula II:
L'-T1-B1-B2-T2 (II)
(In the formula,
L' is absent or is a signal peptide sequence;
T1 is absent or is a tag element;
B1 is a tumor antigen recognition region or a CD3 antigen-binding antibody fragment;
B2 is a CD3 antigen-binding antibody fragment or a tumor antigen recognition region;
T2 is absent or is a tag element;
Each "-" is independently a linker peptide or a peptide bond.
The nucleic acid molecule of claim 1 , 2. The nucleic acid molecule of claim 1 , wherein the EC comprises positions 1 to 104 of the CD3e protein having the amino acid sequence shown in SEQ ID NO:4 and/or the nucleotide sequence shown in SEQ ID NO:3. 4. The nucleic acid molecule of claim 3, wherein C, if present, is a costimulatory signal molecule selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA- 1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof. The nucleic acid molecule of claim 1, wherein C, when present, comprises a 4-1BB-derived costimulatory signal molecule having the amino acid sequence set forth in SEQ ID NO: 10 and/or the nucleotide sequence set forth in SEQ ID NO: 9, and/or a CD28-derived costimulatory signal molecule having the amino acid sequence set forth in SEQ ID NO: 62 and/or the nucleotide sequence set forth in SEQ ID NO : 61 . The nucleic acid molecule of claim 3 , wherein CD3ζ, when present, is a cytoplasmic signaling sequence having the amino acid sequence shown by SEQ ID NO: 12 and/or the nucleotide sequence shown by SEQ ID NO: 11. The nucleic acid molecule of claim 4 , wherein B1 is a tumor antigen recognition region and B2 is a CD3 antigen recognition region. The nucleic acid molecule of claim 4 , wherein the tumor antigen recognition region comprises a receptor or ligand binding domain, an antibody fragment, and/or a T cell receptor (TCR) sequence. The nucleic acid molecule of claim 4 , wherein the tumor antigen recognition region targets the tumor antigen CAIX and/or HER2. The nucleic acid molecule of claim 4 , wherein the tumor antigen recognition region is an antibody fragment having the amino acid sequence shown by SEQ ID NO: 18 and/or the nucleotide sequence shown by SEQ ID NO: 17. The nucleic acid molecule of claim 4 , wherein the tumor antigen recognition region is an antibody fragment that targets HER2 and has the amino acid sequence shown by SEQ ID NO: 66 and/or the nucleotide sequence shown by SEQ ID NO: 65. The nucleic acid molecule of claim 4 , wherein the CD3 antigen-binding antibody fragment comprises a single domain antibody sequence (VHH), a single chain antibody variable region sequence (scFv) and/or an antigen-binding fragment (Fab) that targets CD3. The nucleic acid molecule of claim 4 , wherein the CD3 antigen-binding antibody fragment is derived from an anti- CD3 Ab clone. The nucleic acid molecule of claim 1, comprising the sequence set forth in SEQ ID NO: 73. The nucleic acid molecule of claim 1, comprising the sequence set forth in SEQ ID NO: 67. A vector comprising the nucleic acid molecule of any one of claims 1 to 17 . A genetically modified immune cell that expresses a protein encoded by the nucleic acid molecule of any one of claims 1 to 17 . 20. The genetically modified immune cell of claim 19 , which is a T cell. 21. A non-naturally occurring T cell population comprising the genetically modified immune cells of claim 20 , wherein the T cells are present within the T cell population at a proportion C1 of 10% or greater based on the total number of T cells in the T cell population. 22. A composition comprising: (a) the genetically modified immune cells of claim 20 and/or the T cell population of claim 21 ; and (b) a pharmaceutically acceptable carrier, diluent and/or excipient, wherein the immune cells are T cells . 22. Use of the genetically modified immune cells of claim 20 and/or the T cell population of claim 21 in the preparation of a medicament for the prevention and/or treatment of cancer or tumors , wherein the immune cells are T cells . 24. The use according to claim 23 , wherein the tumor is selected from the following group: hematological tumors, solid tumors, or a combination thereof. 24. The use of claim 23 , wherein the medicament is used in combination with dasatinib.